Project Summary The long-term goals of this project are to understand assembly of repressed chromatin states and to elucidate mechanisms of gene repression in these specialized chromatin environments. Polycomb Group (PcG) proteins interact to form enzyme complexes that modify chromatin and assemble repressed chromatin states in animals, plants, and most fungi. Polycomb Repressive Complex 2 (PRC2) is a highly conserved histone lysine-27 methyltransferase complex that is essential for multicellular development and plays key roles in maintenance of stem cell identify, X-chromosome inactivation, and gene regulation. Mutations that alter PRC2 function are responsible for human Weaver syndrome and multiple cancers. Despite its importance, we lack a clear understanding of the mechanisms that control PRC2 and repress transcription of PRC2 target genes. Preliminary studies with the model fungus Neurospora crassa have begun to address these critical gaps in knowledge. Neurospora is a powerful experimental system that shares key features with higher eukaryotes. Namely, components of PRC2 are structurally and functionally conserved, while they are lacking in both major yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. A targeted RNA-sequencing screen of over 400 N. crassa gene deletion strains that lack known or predicted chromatin-associated or chromatin regulatory proteins uncovered new genes required for PcG repression and are beginning to yield mechanistic insights into their functions. Application of genetic, molecular, proteomic, and genomic approaches will capitalize on these novel findings, and bioinformatic analyses will interrogate this unique RNA-seq data set to: 1) determine how a conserved ATP-dependent chromatin remodeling enzyme controls PRC2 and gene repression at PcG-repressed domains, 2) define mechanisms by which constitutive heterochromatin components regulate PRC2, and 3) define a comprehensive PcG chromatin network in a simple model system. Successful completion of the proposed aims will uncover general principles and mechanisms governing assembly and function of PcG-repressed chromatin domains. Because the first two newly identified components of PcG-mediated silencing are conserved, findings from the proposed studies will serve as a framework for future studies in higher eukaryotes, including humans. In addition, because PcG proteins are linked to cancer and other genetic diseases, understanding the regulation and function of these key chromatin regulators will ultimately improve diagnosis and treatment of human disease.